Optimization of Phenolic Compounds Extraction from Crataegi Fructus

Abstract

Growing interest in the medicinal and nutraceutical uses of hawthorn highlights the need to improve the extraction of bioactive compounds in order to produce high-value products. This study aimed to refine extraction methods to improve the quality and quantity of phenolic compounds, flavonoids, and tannins in Crataegi fructus extracts while preserving their integrity and minimizing the impact of impurities. Phenolic compounds and flavonoids were extracted using ethanol, tannins and water via unconventional ultrasound-assisted extraction protocols. According to the results, significant variations in the total phenolic (TPC), total flavonoid (TFC), and total tannin (TTC) content were observed in correlation with the genotype and the extraction time. The optimal extraction time for TPC and TFC was 150 min of sonication of the samples, while the optimal extraction time for TTC was 30 min of sonication at 99 °C. Ten phenolic compounds, acids (chlorogenic, gallic and syringic acids) and flavonoids (epicatechin, catechin, procyanidin B2, hyperoside, quercetin, isoquercetin and vitexin), were identified in hawthorn fruits extracts by HPLC. Among them, the most abundant were epicatechin, chlorogenic acid, procyanidin B2, catechin and gallic acid. By comparing the maximum contents of phenolic compounds, flavonoids, and tannin extracted from the two species of Crataegi fructus, Crataegus monogyna presented a lower amount of phenolic compounds and tannins, but higher amount of flavonoids, compared to C. pentagyna (1222.15 mg GAE 100 g⁻¹ TPC, 502.47 mg GAE 100 g⁻¹ TTC, and 723.48 mg CE 100 g⁻¹ TFC in C. monogyna vs. 1240.01 mg GAE 100 g⁻¹ TPC, 709.61 mg GAE 100 g⁻¹ TTC, and 549.67 mg CE 100 g⁻¹ TFC in C. pentagyna). Since the climate can influence both the content of bioactive compounds in plants and their extractability, the importance of this study lies in the description for the first time in the literature of hawthorn genotypes selected in Olt County, Romania, in a continental temperate climate with subtropical influences. The results of the study help obtain valuable genotypes for high-quality drugs and food supplements.

1. Introduction

Plants have long been used to treat diseases and support human health, even though the reasons for their effectiveness were not completely understood. Over the last 40 years, more than 60% of the drugs for cancer and infectious diseases have been derived from plants [1]. These include C. monogyna and C. pentagyna, which belong to the genus Crataegus, also known as hawthorn, are widespread in the northern hemisphere, and are used for their medicinal properties and as a food source for their edible parts, including fruits, flowers, and leaves [2]. Due to their longstanding use as traditional medicines and safe food supplements, the European Medicines Agency classified them as “traditional herbal medicinal products” in 2016 [3].

Detailed information on the botany, ethnobotany, and traditional uses of hawthorn can be found in the literature. Several phytochemicals have also been identified and quantified in the genus Crataegus, with studies focusing particularly on flavonoids and anthocyanins, which have been shown to have pharmacological activity. Hawthorn fruits contain large amounts of beneficial secondary metabolites [4,5] with proven therapeutic effects on the respiratory and cardiovascular systems, including coughs, flu, bronchitis, asthma, hypertension, angina pectoris, arrhythmia and congestive heart failure [1,3,6,7]. Polysaccharides, phenolic compounds, flavonoids and anthocyanins are considered the most important bioactive constituents of Crataegus spp. due to their anti-atherosclerotic, antibacterial, anticancer, anti-inflammatory, antispasmodic, cardiotonic, hypotensive, immunobiological and antioxidant properties [1,3,8]. Structure–activity relationships have demonstrated a close correlation between the structure and chemical modifications of these compounds and their biological function [1,3,9]. Furthermore, their bioavailability, biocompatibility, and biodegradability make them promising candidates for applications in the food, medical, and cosmetic industries [1,3].

Plant foods naturally contain phenolic compounds, which have a wide range of complex structures [8,10]. These include phenolic acids, flavonoids, stilbenes, phenolic alcohols, and lignans [1,8]. Although flavonoids, which have a flavone skeleton [11], are not essential for plant survival, they play several roles in plant physiology. They contribute to color and provide protection against reactive oxygen species. They can also act as defensive and/or allelopathic compounds [1,12]. In the animal kingdom, flavonoids act as antimicrobials, stimulate antibody production and exhibit hypotensive and anticarcinogenic properties due to their antioxidant activity [1,3,13].

The American, Chinese and European pharmacopoeias [1,14,15,16,17,18] list species of Crataegus that can be used to produce medicinal products. These pharmacopoeias indicate the minimum concentrations of active constituents (flavonoids and procyanidins) in certain parts of the hawthorn plant that can be used as phytomedicines. For example, hawthorn fruits must contain at least 1% procyanidins, while the leaves and flowers must contain at least 1.5% flavonoids, which are expressed as hyperoside [1]. The main flavonoids found in hawthorn fruit are hyperoside, vitexin, rutin, and their glycosylated derivatives [1,2,5,6,7]. These bioactive compounds, alongside other flavonoids such as quercetin and chlorogenic acid, are believed to contribute to the potential health benefits of hawthorn [1,6]. Therefore, the effective extraction of bioactive compounds from plants is crucial for producing high-value medicinal products

A literature review by Edward et al. (2012) [1] revealed significant variation in the total flavonoid content (TFC) of the fruits, flowers and leaves of various Crataegus species. For example, TFC values for C. Monogyna varied from 4.46 to 147.3 mg 100 g⁻¹ in fruits and from 10.4 to 1026.6 mg 100 g⁻¹ in flowers [1]. These results were influenced by the solvent used for extraction, the extraction time, the temperature, and the particle size of the analyzed sample. All these factors significantly affect solid–liquid extraction [2,19,20,21,22,23]. Therefore, obtaining fruit extracts that are rich in phytochemical compounds is a critical initial step in any chemical analysis or bioactivity assay. As the extraction process is crucial for separating the desired bioactive compounds, the extraction method must be chosen according to the nature of the plant product and compounds to be extracted. Once the method has been selected, the process must be improved by choosing optimal parameters to maximize the quantity of bioactive compounds obtained while minimizing energy and solvent consumption.

Primary and secondary metabolites can be extracted from plants using both classical and unconventional extraction processes. The standard method to non-volatile compounds extraction involves placing the plant in contact with a suitable solvent, such as water, alcohol, acetone, acetonitrile or oil [24]. Phenolic compounds, including flavonoids, are extracted using alcohol or alcohol–water mixtures, whereas polysaccharides and tannins require water only [22,23,24,25,26,27,28,29,30]. Hot water extraction is the most commonly used method for separating polysaccharides and tannins from plants due to its simplicity and convenience. However, this method has several disadvantages, including the risk of denaturing heat-sensitive compounds, long extraction times, and high energy consumption [31,32]. Janicka et al. (2022) [31] demonstrated that increasing the temperature of water under high pressure significantly affects its dielectric constant and, consequently, its solvation power. This enables water to solubilize nonpolar molecules [32]. As water is a green and sustainable solvent, unconventional water-based extraction methods have been developed in recent years. Unlike classical extraction techniques, which can result in the presence of residual solvents or the thermal degradation of bioactive compounds, these processes provide an environmentally friendly alternative for obtaining added-value compounds [31,32,33,34,35]. Recent research has focused on implementing technologies that increase the efficiency of extraction processes by applying intensification techniques such as ultrasound, microwaves, and supercritical fluids [31,32,33,34,35,36,37,38,39]. These techniques are environmentally friendly, using minimal energy and raw materials while producing high yields of various biologically active compounds from different bioresources [22,23,31,32,33,34,35,36,37]. Among them, ultrasound-assisted extraction and green solvent extraction have become increasingly popular [23,31,32,33,34,35,36].

The aim of this study was to refine the extraction methods for phenolic compounds, flavonoids and tannins from Crataegus fruits. The objective was to concentrate these compounds for use in developing innovative products in the future. This would promote a more sustainable economy by facilitating applications in various fields, including the food, pharmaceutical and cosmetic industries. The study aimed to determine the optimal extraction time for these compounds from Crataegi fructus harvested from the Gradinile, Studina and Vladila communes, Olt County, Romania, in order to quantitatively determine their individual and total content. FTIR analysis provided preliminary information on the functional groups associated with major metabolite classes (such as carbohydrates, lipids, and organic acids), supporting the phytochemical characterization of hawthorn genotypes and their potential for nutraceutical or medicinal applications. The results of this study will allow the establishment of the phytochemical profile according to a simplified extraction and identification scheme thanks to the knowledge of the compounds present in hawthorn and the optimal extraction conditions.

2. Materials and Methods

2.1. Chemicals and Reagents

The following chemicals and reagents were used: 96% ethanol, gallic acid, Folin–Ciocalteu reagent, sodium carbonate, sodium nitrite, aluminum chloride, sodium hydroxide and catechin. All of these reagents were sourced from Merck-Sigma-Aldrich in Darmstadt, Germany. The phenolic acids (chlorogenic, syringic, and gallic) and flavonoids (isoquercetin, procyanidin B2, epicatechin, quercetin, and vitexin) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Laboratory Equipment

An ARGOLAB TCN 50 Plus convection oven (Argolab, Prague, Czech Republic) was used to dry the plant material. The following equipment was used to obtain the plant material extracts: a VX-200 Corning-Labnet vortex mixer system (Corning Life Sciences, Tewksbury, MA, USA), an ULTR-2L0-001 Labbox heated ultrasonic bath with an output of 80–100 W at 50–60 kHz (Labbox Labware SL, Migjorn, Spain), and a Labnet Spectrafuge 6c centrifuge (Labnet International Inc., Edison, NJ, USA). Colorimetric determinations were performed using a PerkinElmer Lambda 25 UV-Vis spectrometer (Shelton, CT, USA) and the spectra were processed with UV WinLab, version 2.85 (PerkinElmer Inc., Shelton, CT, USA). Infrared spectra were recorded in the 4000–400 cm⁻¹ region on a Jasco 6300 FTIR spectrometer (Jasco corporation, Hachioji, Tokyo, Japan) equipped with a diamond crystal ATR accessory (PIKE Technologies, Fitchburg, WI, USA), using a TGS detector and cosine apodization. The FTIR spectra were processed with Spectra Manager II, version 2.0 (JASCO Corporation, Hachioji, Tokyo, Japan).

2.3. Material and Preparation

The hawthorn fruits were harvested at consumption maturity from three forest ecosystems belonging to the communes of Gradinile, Studina, and Vladila (43°56′47″ N 24°23′32″ E–44°00′07″ N 24°23′47″ E), Olt County, Romania. The samples were coded with two letters representing the harvesting area (GR, ST or VL), two letters representing the identified species of the genus Crataegus (CM or CP) and a number indicating how many shrubs were used to harvest the fruit from each area. From the genotypes present in the study area, 9 genotypes of red hawthorn (Crataegus monogyna Jacq.) and 2 genotypes of black hawthorn (Crataegus pentagyna Waldst. & Kit.) were analyzed.

The fruits were harvested, washed, and oven-dried at 60–70 °C until constant weight, which was followed by grinding into a fine powder. The dry powders were stored in sealed glass containers in a refrigerator at 4 °C until they were analyzed.

Twelve extracts of each genotype were prepared by placing 1 g of powder in 10 mL of 96% ethanol for the determination of total phenolic compounds and flavonoids and 0.5 g of powder in 10 mL of distilled water for the determination of total tannins. All tubes were vortexed for two minutes using a VX-200 Corning-Labnet Vortex Mixer System. The ethanolic mixtures were then placed in an ultrasonic bath for different extraction times and finally centrifuged at 6000 rpm for 30 min. Immediately after centrifugation, the supernatant of the ethanolic extracts was used for the determination of total phenolic compounds and flavonoids without the need to filter the mixtures. The aqueous mixtures were then incubated in an ultrasonic bath at 99 °C for different extraction times and finally, after incubation of the samples, the supernatants were separated by filtration through a Whatman No. 1 filter and used to determine the total tannin content.

2.4. Experimental Variants

The total phenolic/flavonoid content was determined using the following extraction protocol: vortexing of the samples for 2 min, which was followed by sonication for 90 min (T₉₀), 120 min (T₁₂₀), 150 min (T₁₅₀) or 180 min (T₁₈₀) and finally centrifugation for 30 min.

The total tannin content was determined after vortexing the samples for 2 min, which was followed by sonication at 99 °C for 15 min (T₁₅), 30 min (T₃₀), 45 min (T₄₅), or 60 min (T₆₀) and filtration of the aqueous mixtures.

2.5. The Determination of the Total Phenolic Content (TPC)

The TPC was determined by the spectrometric method, following the methodology described by Giosanu et al. [36], with slight modifications. The ethanolic extract (0.5 mL) was added to a 10 mL glass test tube containing 7 mL distilled water and 0.5 mL Folin–Ciocalteu reagent. After 5 min, 2 mL of 10% sodium carbonate solution was added, and the mixture was incubated for 2 h in the dark at room temperature. A blank sample was prepared from 0.5 mL of Folin–Ciocalteu reagent, 2 mL of 10% sodium carbonate solution, and 7.5 mL of distilled water. The absorbance at 765 nm was measured relative to the blank sample, and the concentration of phenolic compounds was calculated from the calibration curve for gallic acid, which was performed under the same conditions as the samples. The TPC was expressed as mg gallic acid equivalent per 100 g DW (mg GAE 100 g⁻¹ DW).

2.6. Determination of Total Flavonoid Content (TFC)

The TFC was determined using the methodology described by Giura et al. [37] with slight changes. A volume of 0.5 mL ethanolic extract was added to a 10 mL glass test tube containing 6.5 mL distilled water and 0.5 mL 5% sodium nitrate. The mixture was allowed to rest for 5 min, which was followed by the addition of 0.5 mL of 5% aluminum chloride. After 5 min, 2 mL of a 1 M sodium hydroxide solution was added. A blank sample was prepared from 0.5 mL of 5% sodium nitrate, 0.5 mL of 5% aluminum chloride, 2 mL of 1 M sodium hydroxide, and 7 mL of distilled water. The absorbance at 510 nm was measured relative to the blank sample, and the concentration of flavonoids was estimated from the calibration curve for catechin, which was performed under the same conditions as the samples. The TFC was expressed as mg catechin equivalent per 100 g DW (mg CE 100 g⁻¹ DW).

2.7. The Determination of the Total Tannin Content (TTC)

The TTC determination was performed by the spectrometric method according to the methodology described by Giura et al. [37] with slight modifications. The aqueous extract (0.5 mL) was added to a 10 mL glass test tube containing 7 mL distilled water and 0.5 mL Folin–Ciocalteu reagent. After 5 min, 2 mL of 10% sodium carbonate solution was added, and the mixture was incubated for 60 min in the dark at room temperature. A blank sample was prepared from 0.5 mL of Folin–Ciocalteu reagent, 2 mL of 10% sodium carbonate solution, and 7.5 mL of distilled water. The absorbance at 765 nm was measured relative to the blank sample, and the concentration of tannins was estimated using the calibration curve for gallic acid, which was performed under the same conditions as the samples. The TTC was expressed as mg gallic acid equivalent per 100 g DW (mg GAE 100 g⁻¹ DW).

2.8. HPLC-DAD Analysis

In order to identify and quantify the phenolic compounds, the extracts were properly diluted and filtrated using 0.22 μm PVDF syringe filters (Merck Millipore) before the injection, and 10 μL (injection volume) were analyzed using a Shimadzu LC-20AT HPLC system equipped with a quaternary pump, solvent degasser, autosampler, and a photodiode (PDA) detector as described in [28]. The compounds were separated on a Kinetex C18 150 × 4.6 mm, 5 μm column (Phenomenex, Torrance, CA, USA), using mobile phases A (water and phosphoric acid, pH 2.3) and B (acetonitrile) in a gradient mode (between 5 and 90% component B) with a flow rate of 0.8 mL min⁻¹. The total analysis time was 45 min, and the column was maintained at a temperature of 35 °C. The mobile phase was filtered and sonicated in an Elma-Elmasonic P sonication bath to remove air bubbles before starting the HPLC daily run. The following working gradient (expressed in % B) was used: 0–28 min, 5–50% B; 28–38 min, 50–65% B; 38–40 min, 65–30% B; 40–41 min, 30–5% B; 41–45 min, 5% B. Detection was performed in the 200–600 nm range for all peaks, and quantitation was assessed at three specific wavelengths (280, 320, and 360 nm). Calibration curves were created in the concentration range: 0.5–50 μg mL⁻¹, and the concentrations of compounds in the extracts were calculated from the equation obtained for linear regression. Acquisition and processing of the data was performed using LC solution software (Shimadzu, version 1.25). All chemicals and solvents were of analytical or HPLC purity.

2.9. Statistical Analysis

All analyses were performed in triplicate and data were reported as mean (X) ± standard deviation (SD). The results were processed in Microsoft Excel 2010 and IBM SPSS Statistics 26.0 software. One-way ANOVA and a Tukey post hoc test at p < 0.05 were used. Origin Pro 7.0 software was used to fit the experimental data with a second-degree polynomial equation. Jasco Spectra Manager version 2 software was used to process the FTIR spectral data.

3. Results and Discussion

The identification of phenolic compounds was carried out by comparing their retention times with those of authentic standards. The analytical parameters of the HPLC quantitative method for determination of flavonoid compounds are presented in Tables S1 and S2 of the Supplementary Material.

Hyperoside, epicatechin, gallic acid, syringic acid, catechin, procyanidin B2, chlorogenic acid, vitexin, isoquercetin and quercetin were determined in HPLC chromatograms at 280, 320 and 360 nm wavelengths. The chemical profile showed the same major flavonoids and phenolic acids in every sample, and their content is presented in

Table 1. HPLC content of main phenolics in hawthorn (Crataegi fructus) ethanolic extracts (mg phenolic 100 g⁻¹ dry weight).

Genotype	1	2	3	4	5	6	7	8	9	10
GR CM 01	10.67 ± 0.08 h	44.28 ± 0.27 c	1.47 ± 0.01 d	29.80 ± 0.05 c	39.19 ± 0.31 c	146.63 ± 0.06 b	50.24 ± 0.43 c	8.60 ± 0.04 h	2.55 ± 0.01 g	1.54 ± 0.02 d
GR CM 02	9.14 ± 0.07 i	48.71 ± 0.01 b	0.63 ± 0.02 h	14.01 ± 0.01 i	2.80 ± 0.03 j	22.35 ± 0.02 h	21.81 ± 0.01 j	5.69 ± 0.01 k	1.57 ± 0.01 j	1.06 ± 0.01 h
GR CM 03	11.91 ± 0.11 f	55.52 ± 0.42 a	1.65 ± 0.04 c	19.61 ± 0.15 f	3.93 ± 0.03 i	11.63 ± 0.08 j	29.10 ± 0.35 h	9.66 ± 0.14 g	3.86 ± 0.04 d	0.97 ± 0.01 i
ST CM 01	43.29 ± 0.32 b	31.46 ± 0.27 d	0.90 ± 0.02 e	33.99 ± 0.17 a	44.95 ± 0.27 b	138.05 ± 0.35 c	42.97 ± 0.33 e	34.44 ± 0.21 c	5.51 ± 0.04 b	2.08 ± 0.01 b
ST CM 02	19.96 ± 0.01 e	12.90 ± 0.01 i	3.48 ± 0.01 a	18.07 ± 0.03 g	15.91 ± 0.04 d	78.01 ± 0.12 e	29.76 ± 0.06 g	17.14 ± 0.03 f	3.81 ± 0.02	1.74 ± 0.03 c
ST CM 03	48.05 ± 0.02 a	28.33 ± 0.07 f	2.19 ± 0.04 b	31.95 ± 0.28 b	58.29 ± 0.01 a	187.95 ± 0.14 a	57.12 ± 0.08 b	37.73 ± 0.12 b	5.13 ± 0.02 c	1.25 ± 0.01 e
VL CM 01	11.35 ± 0.13 g	28.48 ± 0.18 f	0.74 ± 0.08 g	14.73 ± 0.07 h	11.43 ± 0.10 f	82.97 ± 0.58 d	28.55 ± 0.21 i	6.77 ± 0.06 i	1.36 ± 0.01 k	1.14 ± 0.01 g
VL CM 02	11.89 ± 0.02 f	17.72 ± 0.03 h	0.21 ± 0.02 i	7.02 ± 0.07 k	4.82 ± 0.13 h	27.68 ± 0.26 g	14.65 ± 0.06 k	6.08 ± 0.19 j	1.84 ± 0.01 i	1.19 ± 0.01 f
VL CM 03	26.88 ± 0.04 c	8.59 ± 0.01 j	0.83 ± 0.01 f	13.02 ± 0.09 j	13.28 ± 0.01 e	44.06 ± 0.10 f	34.97 ± 0.19 f	19.80 ± 0.03 e	9.05 ± 0.02 a	3.00 ± 0.01 a
GR CP 01	20.65 ± 0.09 d	20.64 ± 0.11 g	1.63 ± 0.03 c	23.02 ± 0.20 d	9.93 ± 0.16 g	21.82 ± 0.09 i	94.46 ± 0.07 a	54.72 ± 0.09 a	2.44 ± 0.02 h	0.36 ± 0.01 k
ST CP 01	10.60 ± 0.03 h	29.26 ± 0.20 e	0.72 ± 0.01 g	22.33 ± 0.05 e	4.04 ± 0.06 i	22.50 ± 0.04 h	44.19 ± 0.15 d	25.04 ± 0.07 d	3.07 ± 0.02 f	0.41 ± 0.01 j
Mean	20.40	29.63	1.31	20.69	18.96	71.24	40.71	20.52	3.65	1.34
Median	11.93	28.47	0.89	19.61	11.42	44.07	34.98	17.14	3.08	1.19
Standard Deviation	13.26	14.36	0.89	8.27	18.68	59.17	21.08	15.52	2.18	0.73
Minimum	9.07	8.58	0.19	6.96	2.77	11.56	14.59	5.68	1.35	0.36
Maximum	48.07	55.94	3.49	34.15	58.29	188.08	94.53	54.81	9.07	3.01

Results are expressed as mean ± standard deviation (n = 3). Compounds identification: 1—Hyperoside, 2—Gallic acid, 3—Syringic acid, 4—(+)-Catechin, 5—Procyanidin B2, 6—(−)-Epicatechin, 7—Chlorogenic acid, 8—Isoquercetin, 9—Quercetin, 10—Vitexin. Different letters as exponent indicate statistically significant differences (Tukey test, p < 0.05).

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Representative chromatograms illustrating the presence of phenolic acids and flavonoids in the hawthorn extracts along with standard solution mixture are presented in Figures S1 and S2 of the Supplementary Material.

In previous studies of various Crataegus species, phenolic acids such as chlorogenic, syringic and gallic [7,40] have been identified and quantified along with flavonoids such as epicatechin, catechin, quercetin, and procyanidin B2 [41,42,43,44]. The results of previous studies correlate with the present results, as shown in Table 1. Standard solution mixture and different genotypes extracts are presented in Figure 1, where two predominant compounds were identified based on a combination of retention time and spectral matching. Epicatechin was the major compound in seven out of eleven Crataegus fruit extract samples, accounting for 25.4–44.2% of the total identified phenolic compounds. The next two phenolic compounds most found in all of the investigated samples are chlorogenic and gallic acid (23.21% of the total identified phenolic compounds) followed by catechin and hyperoside (20.14% of the total identified phenolic compounds). The high level of epicatechin in the samples may be correlated further to the well-known antioxidant and antitumoral activity of the hawthorn extracts, since multiple studies proved that extracts rich in epicatechin inhibit the proliferation of cancer cells [5,13]. According to a previous study by Alirezalu et al. [7], the C. monogyna fruit extracts contain approximately 0.40 mg g⁻¹ of chlorogenic acid, which is a quantity that is similar to the ones obtained in this study (between 14.65 and 57.12 mg 100 g⁻¹ DW in VL CM 02 and ST CM 03). The highest amount of chlorogenic acid was found in the Crataegus pentagyna sample (GR CP 01), namely 94.46 mg 100 g⁻¹ DW along with the highest isoquercetin concentration around 54.72 mg 100 g⁻¹ DW. For chlorogenic acid, gallic acid, isoquercetin and procyanidine B2, the concentrations were almost double for the same sample species (C. pentagyna) but from different location areas (GR CP 01 compared to ST CP 01). Because hyperoside has many bioactive properties such as scavenging reactive oxygen species, preventing free radical induced oxidation and increasing superoxide dismutase activity, its highest amount in the fruit extracts obtained in the samples from the ST region (between 19.96 ± 0.01 and 48.05 ± 0.03 mg 100 g⁻¹ DW) enhanced the extracts’ beneficial potential along with two other predominant compounds, procyanidin B2 and epicatechin. Although syringic acid was known to be present in hawthorn extracts, in this study, the amount of this compound was insignificant, and other bioactive components such as vitexin and quercetin content were in very low amounts.

By comparing the species Crataegus monogyna and Crataegus pentagyna from the chemical profile and phenolic content point of view, the C. pentagyna extracts had the lowest content in phytochemical constituents, and in a completely opposite way, the samples from the ST region were of lower quality (total phenolic content) than the samples from the GR region. For the C. monogyna species, the samples from the ST region contain higher amounts of phenolic compounds, particularly hyperoside, procyanidine B2, epicatechin and chlorogenic acid, followed by the samples from the GR region, while the samples with the lowest phenolic content among all the analyzed extracts were from the VL region. A positive correlation between individual phenolic compounds determination and total phenolic/flavonoid content (TPC/TFC) was observed regarding the species from different regions (GR, ST, and VL). The hawthorn extracts from the ST region had the most effective antioxidative constituents followed by hawthorn species from the GR and VL regions. The variability in the reported phenolic compound contents and flavonoid concentrations within one species could be related to differences in several environmental factors such as growth conditions (soil moisture, light, temperatures) and genetic background [5,27].

From the total phenolic compounds determined spectrophotometrically, the largest proportion is represented by (−)-epicatechin (on average 5.81%), followed by chlorogenic (3.32%) and gallic (2.42%) acids, at the opposite pole being vitexin, representing 0.11% of TPC. Compared to C. monogyna, the highest percentages of chlorogenic acid and isoquercetin were found in a genotype belonging to the species C. pentagyna, GR CP 01 (8.15% and 4.72%). Reported at the TPC, four representatives of C. monogyna stood out. ST CM 03 had the highest percentage of hyperoside (4.00%), (+)-catechin (2.66%), procyanidin B2 (4.85%), and (−)-epicatechin (15.65%), GR CM 03 presented the highest percentage of gallic acid (4.71%), ST CM 02 contained 0.28% syringic acid, and VL CM 03 presented 0.68% quercetin and 0.22% vitexin.

Spectrometric analysis was performed to determine the total phenolic (TPC) and total flavonoid (TFC) content of the extracts obtained from 11 Crataegus genotypes (

Table 2. Variation in TPC (mg GAE 100 g⁻¹ DW) in ethanolic extracts of the hawthorn genotypes at different extraction times.

Genotype	T90	T120	T150	T180
GR CM 01	968.47 ± 0.59 e	1150.49 ± 0.44 f	1167.73 ± 0.59 h	1138.88 ± 0.10 g
GR CM 02	936.83 ± 0.66 f	1036.23 ± 9.10 i	1042.70 ± 0.88 j	1033.70 ± 0.06 k
GR CM 03	992.31 ± 0.67 d	1073.49 ± 0.12 h	1178.86 ± 0.68 g	1050.50 ± 0.21 j
ST CM 01	1032.45 ± 0.84 a	1365.53 ± 2.31 a	1399.11 ± 0.12 a	1258.94 ± 0.46 c
ST CM 02	1009.93 ± 0.17 c	1086.95 ± 0.42 g	1236.69 ± 1.00 e	1172.31 ± 0.18 e
ST CM 03	1028.87 ± 0.34 b	1173.42 ± 0.08 e	1200.92 ± 1.08 f	1155.89 ± 0.06 f
VL CM 01	903.06 ± 0.96 g	1012.29 ± 0.41 k	1181.00 ± 0.13 g	1088.41 ± 2.14 i
VL CM 02	854.13 ± 0.35 h	1175.62 ± 0.67 d	1260.51 ± 0.29 d	1202.87 ± 0.33 d
VL CM 03	903.95 ± 0.78 g	1286.78 ± 0.74 b	1334.77 ± 3.42 b	1281.49 ± 0.31 a
GR CP 01	721.79 ± 0.34 j	1023.51 ± 0.42 j	1158.82 ± 0.02 i	1129.11 ± 0.07 h
ST CP 01	774.14 ± 0.34 i	1196.67 ± 0.59 c	1321.16 ± 0.93 c	1268.16 ± 0.02 b
All genotypes	920.58 ± 99.54	1143.42 ± 108.36	1225.40 ± 95.83	1161.58 ± 82.54

Mean ± standard deviation (n = 3). Different letters as exponent indicate statistically significant differences between genotypes (Tukey test, p < 0.05).

and Figure S3 for TPC,

Table 3. Variations in TFC (mg CE 100 g⁻¹ DW) in ethanolic extracts of the hawthorn genotypes at different extraction times.

Genotype	T90	T120	T150	T180
GR CM 01	580.74 ± 2.82 a	719.14 ± 0.39 c	871.13 ± 2.19 a	822.11 ± 5.87 a
GR CM 02	262.39 ± 0.08 k	451.33 ± 0.79 j	551.61 ± 1.53 j	519.58 ± 2.18 i
GR CM 03	424.22 ± 0.49 f	709.13 ± 0.32 e	729.32 ± 2.65 e	681.86 ± 1.53 e
ST CM 01	552.41 ± 2.60 c	756.26 ± 2.00 b	844.47 ± 1.15 b	744.89 ± 0.30 c
ST CM 02	436.14 ± 0.37 e	712.98 ± 0.11 d	734.04 ± 0.69 d	713.49 ± 2.14 d
ST CM 03	562.37 ± 1.42 b	792.70 ± 0.70 a	812.44 ± 0.18 c	770.81 ± 3.64 b
VL CM 01	356.05 ± 0.22 h	635.86 ± 0.54 f	685.60 ± 1.04 f	652.44 ± 1.76 f
VL CM 02	532.83 ± 0.71 d	571.55 ± 1.49 h	602.90 ± 0.39 i	596.13 ± 1.21 g
VL CM 03	376.40 ± 0.39 g	584.91 ± 1.97 g	640.94 ± 0.53 h	588.17 ± 0.98 h
GR CP 01	289.49 ± 0.34 j	393.07 ± 0.60 k	453.92 ± 1.28 k	432.20 ± 1.66 j
ST CP 01	337.93 ± 0.49 i	563.24 ± 2.15 i	645.42 ± 1.23 g	593.63 ± 1.87 gh
All genotypes	428.27 ± 110.61	626.38 ± 123.00	688.34 ± 123.24	646.85 ± 111.48

Mean ± standard deviation (n = 3). Different letters as exponent indicate statistically significant differences between genotypes (Tukey test, p < 0.05).

and Figure S6 for TFC), which were responsible for the fruit therapeutic value. Following statistical processing of the obtained data using one-way ANOVA, significant differences were found between the genotypes and extraction times for TPC and TFC (p < 0.05), as evidenced by the results of the Duncan multiple range tests.

According to Enescu et al. [45], ethanolic extracts obtained by the ultrasound-assisted extraction method had a major influence on the TPC and TFC in fruits. Bamba et al. [46] mentioned that due to the diversity of phenolic compounds and the biological matrices in which they are incorporated, extracts of these compounds cannot be easily generalized or standardized by the ultrasound-assisted method. For TPC, determinations made after 90 min of extraction (T₉₀) recorded the lowest amounts compared to the other extraction times (Figure S3) with a total average of only 920.58 mg GAE 100 g⁻¹ (Table 2). The genotype with the lowest amount was GR CP 01 (black hawthorn) at 721.79 mg GAE 100 g⁻¹. ST CM 01 (red hawthorn) had the highest content at 1032.45 mg GAE 100 g⁻¹.

Within C. monogyna, the lowest value was 854.13 mg GAE 100 g⁻¹ (VL CM 02). For T₁₂₀, TPC values started to increase for all genotypes with a total average of 1143.42 mg GAE 100 g⁻¹. An extraction time of 150 min (T₁₅₀) was found to produce the highest total phenolic content (TPC) values for all genotypes with an average value of 1225.4 mg GAE 100 g⁻¹. For the T₁₈₀ extraction time, there was a slight decrease in TPC compared to the previous time, the mean of all genotypes being 1161.58 mg GAE 100 g⁻¹.

The results obtained in this study are in agreement with the literature. In a previous study, Enescu et al. [45] found that for extracts obtained from chokeberry pomace, the TPC increased with extraction time and identified an optimal result at 150 min. Similar results are reported by Bamba et al. [46], which found that for sonication times of 30 and 60 min, both polyphenolic and phenolic compounds increase with extraction time.

The concentration of extracted phenolic compounds increased significantly: on average by 25.41% with the extension of extraction time from T₉₀ to T₁₂₀ and by 34.74% from T₉₀ to T₁₅₀ (Figure S4). Extending the extraction time to 180 min (T₁₈₀) resulted in a significant average reduction in the extracted phenolic content compared to the previous extraction time.

Comparing the two hawthorn species (Figure S5), it is noted that the largest (and also significant) increase in extracted phenolic content resulted from extending the extraction time in C. pentagyna species from T₉₀ to T₁₅₀, respectively by 65.56%, compared to 48.09% from T₉₀ to T₁₂₀. In C. monogyna, although in a small percent, the increase in phenolic content was also significant between T₉₀ and T₁₅₀. The results for TPC at T₁₈₀ were significantly lower compared to those at T₁₅₀ in C. pentagyna but comparable to those at T₁₂₀ in C. monogyna species. In addition, in ST CM 02, VL CM 01, and VL CM 02, unlike the other C. monogyna genotypes, it was observed that the level of TPCs determined at T₁₈₀ was higher than that determined at T₁₂₀. The same type of treatment resulted in a reduction in the total extracted phenolic content of 2.6% and 4.0%, respectively, in C. pentagyna. However, the TPC levels determined at T₁₈₀ were higher than those determined at T₁₂₀.

Chandini et al. [47] noted in a study conducted on an aqueous extract of black tea leaves (Camellia sinensis) that at intervals ranging from 10 to 120 min, the optimal extraction time was 40 min and that extending the extraction period led to a decrease in the amount of TPC, which is attributed to the variation in molecular weight of bioactive compounds. Jovanović et al. [48] reported similar conclusions for ethanolic extracts of wild thyme (Thymus serpyllum), with an obvious decrease in TPC from 15 min of extraction to 30 min of extraction, by exposure to an extended time in sonication and high temperatures. The amounts of TPC reported were much lower than those determined in the present study for both the research conducted by Jovanović et al. [48], as well as for the research carried out on fruits mentioned previously, which indicates that the fruits of the genus Crataegus have a much richer phenolic content than other species. In another study [49], it was noted that hydrolysis and oxidation are the main phenomena that can cause a decrease in the extracted phenolic compounds quantity by increasing the extraction time. A study conducted on wild fruits in Hungary by Sik et al. [50] found an increase in phenolic content for the wild apple species (Malus sylvestris) of 41.5% between extraction times at 30 and 90 min, while for plum (Prunus domestica subsp. italica) and quince (Cydonia oblonga) genotypes, there was an increase in the same range of only 8.49%, with obvious differences between wild fruits even between species of the same family, as is the case in this study.

For TFC, the same extraction times were maintained as for TPC (Figure S6), and the behavior of the extracts was similar. T₉₀ led to a reduction in TFC, resulting in a total average of 428.27 mg CE 100 g⁻¹ (Table 3). T₁₂₀ showed an increase in TFC by an average of 626.38 mg CE 100 g⁻¹. The maximum TFC was also identified at T₁₅₀ sonication time with an average value increasing up to 688.34 mg CE 100 g⁻¹. T₁₈₀ produced a slight decrease in TFC with an average value of 646.85 mg CE 100 g⁻¹.

The TFC represented on average 56.17% of the TPC. Among the two species, C monogyna had the highest percentage of TFC reported to TPC content (approx. 59% TFC, compared to C. pentagyna, with 44.01% TFC). The genotype with the highest percentage of TFC was GRCM01 (74% of 1167.73 mg GAE 100⁻¹ g TPC). Four genotypes had a TFC percentage greater than 60%, three exceeded 50%, three contained TFC above 40% and only one was above 30%.

Enescu et al. [45] reported for TFC an optimal extraction time of 150 min for chokeberry pomace, mentioning that extending the time beyond this limit may result in the extraction of bioactive compounds other than phenolic ones. Arancibia-Avila et al. [51] also mentioned that extended heat treatment on extracts can lead to a decrease in both TFC and TPC. The total flavonoid content (Figure S7) increased, similarly to phenolic compounds, with the extension of extraction time from T₉₀ to T₁₂₀, on average by 49.8%, reaching the maximum in T₁₅₀ (65.6%). T₁₈₀ led without exception to a reduction in TFC compared to T₁₅₀, but it was higher than that determined at T₁₂₀. The decrease in phenolic content after a longer extraction time was also found by Chandini et al. [47], which explains this behavior by the catechin condensation process. Jovanović et al. [48] observed the same trend for TFC and TPC, which was in accordance with the results from this study, but also noted that besides time, other factors can influence the extraction of bioactive compounds, such as the particle size of the plant material and the solid–liquid ratio. Their study also concluded that ethanolic concentration did not show significant differences in phenolic compounds contents. Data reported by Sik et al. [50], for extracts obtained from wild fruits, showed a preference for ethanolic extracts over methanol due to the possibility of further use in food industry and established that prolonged extraction times led to the degradation of phenolics in fruit extracts.

An analysis of each of the two hawthorn species (Figure S8) also indicated that T₁₂₀ determinations resulted in average increases in flavonoid content of 49.5% for C. monogyna and 51.2% for C. pentagyna. When the extraction process was extended from T₉₀ to T₁₅₀, the flavonoid content was only insignificantly higher in C. monogyna (63.8%) and significantly higher in C. pentagyna (73.9%). In both species, further analysis of the extract at T₁₈₀ showed insignificantly lower flavonoid amounts compared to T₁₅₀. Comparing the flavonoids content obtained by extending T₉₀ to T₁₂₀ in C. monogyna genotypes, an increase from 7.3% (VL CM 02) to 78.6% (VL CM 01) was observed. The highest increase in TFC for extracts was observed especially in the GR CM 02 genotype (110.2% more TFC extracted) as well as in GR CM 01 (with an additional 50.0% TFC) when comparing the extraction times T₉₀ and T₁₅₀ to T₁₂₀. By contrast, ST CM 03, GR CM 03, and ST CM 02 showed the lowest increases in TFC. The extraction methods and genotype significantly influenced the flavonoid compositions, which was an aspect also mentioned in other studies [52,53,54].

Regarding the TTC values (Figure S9), it was evident that a period over 30 min of extraction led to the degradation of tannins. Thus, the optimal extraction time identified for TTC was T₃₀ when a maximum TTC of 733.65 mg GAE 100 g⁻¹ (

Table 4. Variations in TTC (mg GAE 100 g⁻¹ DW) in water extracts of hawthorn genotypes at different extraction times.

Genotype	T15	T30	T45	T60
GR CM 01	200.02 ± 0.61 k	336.74 ± 0.12 k	273.23 ± 0.90 k	261.13 ± 0.62 i
GR CM 02	362.24 ± 0.75 f	476.70 ± 0.38 g	425.19 ± 0.82 h	217.84 ± 0.25 k
GR CM 03	283.29 ± 0.56 i	409.27 ± 0.25 j	383.89 ± 0.30 j	270.31 ± 0.09 h
ST CM 01	382.96 ± 0.64 e	557.69 ± 0.66 e	477.58 ± 0.59 f	292.59 ± 0.58 f
ST CM 02	345.36 ± 0.40 h	499.33 ± 0.38 f	488.28 ± 0.65 e	286.93 ± 0.40 g
ST CM 03	258.46 ± 0.69 j	457.93 ± 0.61 i	397.88 ± 0.34 i	233.56 ± 0.49 j
VL CM 01	353.12 ± 0.37 g	467.92 ± 0.27 h	430.97 ± 0.93 g	384.51 ± 0.74 e
VL CM 02	510.93 ± 0.34 a	668.35 ± 0.33 c	609.80 ± 0.06 c	439.81 ± 0.56 d
VL CM 03	494.22 ± 0.16 c	648.31 ± 0.21 d	565.44 ± 0.37 d	480.12 ± 0.27 c
GR CP 01	502.93 ± 0.30 b	733.65 ± 0.63 a	713.77 ± 0.73 a	549.05 ± 0.25 b
ST CP 01	401.91 ± 0.38 d	685.57 ± 0.69 b	682.14 ± 0.59 b	607.00 ± 0.32 a
All genotypes	372.31 ± 98.69	540.13 ± 123.69	495.29 ± 130.37	365.71 ± 130.51

Mean ± standard deviation (n = 3). Different letters as exponent indicate statistically significant differences between genotypes (Tukey test, p < 0.05).

) was recorded for C. pentagyna species (GR CP 01) and 668.35 mg GAE 100 g⁻¹ for C. monogyna species (VL CM 02). With a total average of 365.71 mg GAE 100 g⁻¹, the TTC value at T₆₀ was lower compared to T₁₅ (372.31 mg GAE 100 g⁻¹), although in some cases at T₁₅, lower values were recorded for some genotypes such as GR CM 01, ST CP 01 or VL CM 01 compared to T₆₀. Regarding T₃₀, a significant increase was noted compared to T₁₅, which was followed by slight decreases in TTC at T₄₅ with an average of 495.29 mg GAE 100 g⁻¹ compared to the previous T₃₀ extraction time that averaged 540.13 mg GAE 100 g⁻¹.

Unlike phenolic compounds and flavonoids, sonication for 30 min favored the extraction of the highest amounts of tannin, which was on average 47.52% more than 15 min sonication (T₁₅). By extending the duration of the extraction process to 45 min, a reduction in the extraction efficiency was observed (34.27% TTC compared to T₁₅), while increasing the duration of the process to 60 min led to a decrease in the extracted tannins even compared to T₁₅ (Figure S10).

Among the nine genotypes of C. monogyna, seven showed a drastic reduction (below the amount obtained at T₁₅) of TTC as a result of 60 min of sonication. The most intensively affected was GR CM 02 with a loss of approximately 40% of its tannin content. This aspect explains the differences between the genotypes regarding the effect of the extraction time (Figure S11). In addition, it could be observed that sonication for 30 min produced the highest increases in TTC extracted precisely in the cultivars where the first extraction time led to the lowest concentrations (GR CM 01 with 200.06 mg GAE 100 g⁻¹ and ST CM 03 with 258.46 mg GAE 100 g⁻¹).

The tannin content represented on average 44.08% of that of TPC. Unlike TFC, in this case, the highest percentage of TTC compared to TPC was found in C. pentagyna (57.60% vs. 40.98% in C. monogyna). The C. pentagyna GR CP 01 presented the highest percentage of TTC from TPC (63.31%), while among C. monogyna, VL CM 02 had 53.02% TTC from TPC and was followed by ST CP 01 with 51.89% TTC. Three genotypes presented a TTC percentage of over 40%, and three exceeded 30%. The lowest percentage of TTC was determined in GR CM 01 (28.84%), which was the genotype with the highest percentage of flavonoids.

This study found that on average, the highest amounts of phenolic compounds, flavonoids (determined after 150 min of sonication), and tannins (determined after 30 min of sonication) obtained in the eleven hawthorn genotypes selected from Olt County were 1225.40 mg GAE mg⁻¹ TPC, 691.88 mg CE 100 g⁻¹ TFC, and 540.13 mg GAE 100 g⁻¹ TTC. The analysis of the maximum contents of phenolic compounds, flavonoids, and tannin extracted depending on the hawthorn species indicated that on average, C. monogyna presented a lower content of phenolic compounds and tannins but higher content of flavonoids compared to C. pentagyna (1222.15 mg GAE 100 g⁻¹ TPC, 502.47 mg GAE 100 g⁻¹ TTC, and 723.48 mg CE 100 g⁻¹ TFC in C. monogyna vs. 1240.01 mg GAE 100 g⁻¹ TPC, 709.61 mg GAE 100 g⁻¹ TTC, and 549.67 mg CE 100 g⁻¹ TFC in C. pentagyna).

The close correlation between total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC) with extraction times was clearly demonstrated through both the polynomial equations and the regression coefficients presented in

Table 5. Polynomial equations and correlation coefficients (R²) to test the influence of extraction time (x) on total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC).

Genotype	Polynomial Equation	R2
GR CM 01	TPC = −1.6221 + 15.2944 x − 0.0497 x2	0.9992
	TFC = −6.1267 + 9.0964 x − 0.0254 x2	0.9939
	TTC = −2.6514 + 18.3407 x − 0.2617 x2	0.9815
GR CM 02	TPC = 2.2876 + 14.7186 x − 0.0502 x2	0.9993
	TFC = −5.9149 + 5.2737 x − 0.0126 x2	0.9959
	TTC = 10.2189 + 28.1838 x − 0.4143 x2	0.9938
GR CM 03	TPC = 2.8052 + 16.1058 x − 0.0572 x2	0.9995
	TFC = −23.2279 + 8.6563 x − 0.0255 x2	0.9913
	TTC = 6.0937 + 22.2767 x − 0.3001 x2	0.9951
ST CM 01	TPC = −23.5601 + 18.9416 x − 0.0646 x2	0.9958
	TFC = −23.3852 + 9.6008 x − 0.0271 x2	0.9924
	TTC = 7.5269 + 31.0223 x − 0.4415 x2	0.9913
ST CM 02	TPC = −1.9259 + 15.4288 x − 0.0492 x2	0.9978
	TFC = −2.0688 + 8.8739 x − 0.0268 x2	0.9924
	TTC = 0.8469 + 28.7488 x − 0.3995 x2	0.9997
ST CM 03	TPC = −1.9588 + 16.3405 x − 0.0551 x2	0.9998
	TFC = −7.1126 + 9.0923 x − 0.0257 x2	0.9909
	TTC = −9.6106 + 25.0927 x − 0.3508 x2	0.9841
VL CM 01	TPC = 3.4655 + 13.8322 x − 0.0433 x2	0.9988
	TFC = −10.8491 + 7.8683 x − 0.0226 x2	0.9931
	TTC = 22.0857 + 23.7584 x − 0.3019 x2	0.9627
VL CM 02	TPC = −17.7699 + 15.1208 x − 0.0454 x2	0.9968
	TFC = 15.1336 + 8.2341 x − 0.0283 x2	0.9945
	TTC = 24.6786 + 36.5768 x − 0.5009 x2	0.9789
VL CM 03	TPC = −22.8064 + 16.0956 x − 0.0481 x2	0.9964
	TFC = 5.0979 + 7.5411 x − 0.0231 x2	0.9977
	TTC = 31.8917 + 33.4677 x − 0.4432 x2	0.9547
GR CP 01	TPC = −29.7164 + 12.7193 x − 0.0353 x2	0.9882
	TFC = −3.0019 + 5.4754 x − 0.0173 x2	0.9948
	TTC = 11.5349 + 38.9339 x − 0.5035 x2	0.9951
ST CP 01	TPC = −26.8505 + 13.8338 x − 0.0372 x2	0.9959
	TFC = 1.5791 + 7.1749 x − 0.0213 x2	0.9969
	TTC = −0.8357 + 33.6035 x − 0.3941 x2	0.9931

as well as the graphical representations shown in Figure 2. These analytical tools highlight a significant trend.

The TPC, TFC and TTC values increased consistently as the extraction time progressed, reaching a peak before declining gradually. This slow decline underscores the dynamic equilibrium that occurs during the extraction process with a few notable exceptions observed in the TFC measurements. These deviations may indicate the specific chemical properties or stability factors of phenolic compounds, particularly flavonoids and tannins, when exposed to extraction conditions over a prolonged period. Such findings are in strong agreement with the experimental data and other research showing that extraction is influenced by various factors, such as time, temperature, solvent and concentration [55,56,57]. The results suggest that obtaining an accurate determination of total phenolic compounds, flavonoids and tannins requires a carefully optimized extraction protocol. Specifically, extending the extraction time to an optimal duration is crucial for maximizing the yield of these bioactive compounds. However, the decline observed with longer extraction times also highlights the importance of identifying the tipping point at which further extraction no longer provides additional benefit and may actually lead to degradation or loss of the bioactive compound.

Figure 3 shows the absorption spectra of powdered hawthorn fruit in the range 4000–400 cm⁻¹. The spectra exhibit distinctive peaks. The region of 3400–3200 cm⁻¹ indicates a symmetric (sym) and asymmetric (asym) stretching of polymeric hydroxyl group (O–H), H-bonded stretching, which is characteristic of polyphenolic compounds. In the region of 2960–2854 cm⁻¹, the –CH, –CH₂ and –CH₃ asymmetric and symmetric stretching vibrations, derived from carbohydrates and sugars in dried hawthorn genotypes, can be observed. The stretching of the C–H and C=C–C aromatic bond appears in the region of 1615–1556 cm⁻¹. The phenolic C–O stretching was detected at around 1200 cm⁻¹. This stretching is due to the C–O of pyran, which is typical of flavonoid C-rings. These group frequencies are closely associated with the existence of aromatic chemicals. Differences in the band intensity and position between genotypes align with the quantitative differences observed in HPLC and spectrophotometric analyses.

The FTIR analysis confirmed the presence of functional groups corresponding to key metabolite classes such as carbohydrates, lipids, and organic acids [58,59,60,61,62], which are known to contribute to the medicinal and nutritional value of hawthorn. The differences observed between genotypes in the FTIR spectra (Table S3) are consistent with the quantitative variability found through HPLC and spectrophotometric analyses, suggesting a strong genotype-dependent phytochemical profile.

A comparative study of hawthorn species harvested from the communes of Gradinile, Studina, and Vladila in Olt County, Romania, will be carried out in the future. The analyzed parameters will be correlated with antioxidant activity, which will be evaluated using various methods. At the same time, the extraction efficiency of different solvents or solvent mixtures will be compared with the extraction yields and kinetics.

4. Conclusions

Ultrasound-assisted extraction is an efficient and economical extraction technique. In this study, it was used to extract bioactive compounds from Crataegi fructus. The efficacy of the extraction process was evaluated based on the total phenolic (TPC), total flavonoid (TFC) and total tannin (TTC) content. The search for optimal extraction conditions ensures that bioactive compounds are not degraded during extraction, which is a key requirement for the development of new products. In this study, the optimal extraction time for phenolic compounds and flavonoids was 150 min of sonication of the samples, while the optimal extraction time for tannins was 30 min of sonication at 99 °C. The results determined in this study indicate the potential exploitation of 11 hawthorn genotypes for medicinal and nutraceutical purposes. In addition to extraction time, various factors can affect the extraction of TPC, TFC and TTC, including the solid–liquid ratio, particle size, pH level and temperature. Further research into these valuable wild fruit resources is required. Therefore, an evaluation of hawthorn genetic resources could provide valuable data for screening genotypes with high bioactive content in order to produce nutraceutical and food supplements rich in phytochemical compounds.